Bilirubin is a yellow substance which is formed in the blood by degradation of hemoglobin. The rapid and accurate detection of bilirubin in blood serum is vitally important to medical diagnosis of disease states, e.g., jaundice, in human beings.
The present invention provides new compositions and methods for the analysis of bilirubin. The invention provides enzyme preparations which unexpectedly react specifically with bilirubin and cause color changes through which one can detect and measure bilirubin.
To applicant's knowledge, there is little discussion in the literature of enzyme preparations exhibiting bilirubin activity, especially of fungal enzyme preparations which both degrade bilirubin and produce hydrogen peroxide. H. Plieninger and L. Petzold, in Z. Physiol. Chem., 297:238, 1954 describe what they refer to as "mushroom enzymes" having activity on bilirubin. The enzymatic activity is reportedly obtained merely by incubating mushroom juice from the mushroom Psalliota campestris (now known as Agaricus bisporus or Agaricus campestris) with bilirubin. Plieninger et al mention no hydrogen peroxide generation. Plieninger et al base their findings solely on observation of oxygen uptake when a bilirubin medium was incubated with mushroom juice. They reported no oxygen uptake upon incubation of the bilirubin medium by itself. However, they failed to provide any control to measure oxygen uptake in the absence of bilirubin. Thus, it is possible that the oxygen uptake was caused by oxidation of one or more of the many possible unknown components in their crude, unpurified mushroom juice, rather than by enzymatic activity.
Furthermore, the reaction time scale reported by Plieninger et al in terms of hours is highly atypical of enzyme catalyzed reactions, which characteristically occur within minutes or less. In any case, as the comparative data in appended Example 1 show, applicant has found no evidence to substantiate the claim of Plieninger et al that simple mushroom juice exhibits enzymatic activity on bilirubin.
R. Brodersen and P. Bartels, in Europ. J. Biochem., 10:468, 1969 describe an insoluble "bilirubin oxidase" isolated from guinea pig brain. They report that the insoluble enzyme converts bilirubin to a spectrum of products, including a material showing light absorption maxima at 375 nm and at 630 through 650 nm, which suggests the presence of biliverdin. They report no generation of hydrogen peroxide. (See pages 472 and 473 as well as FIG. 4 of the Brodersen et al article.)
Prior to the present invention, no bilirubin assay procedure known to applicant employed an enzymatic determination specific for bilirubin. Presumably, this is because of the small number of known enzyme preparations with specific activity for bilirubin. The most widely used assays for bilirubin are based upon the so-called diazo method or one of its many variants. This technique employs a coupling reaction of bilirubin with a diazonium salt, such as diazosulfanilic acid, to form a pigment having an extinction coefficient higher than that of bilirubin. The diazo method has a variety of problems. For example, as noted in Clinical Chemistry-Principles and Technics, edited by R. J. Henry, D. C. Cannon, and J. W. Winkelman, Harper and Row Publishers, 2nd Edition, pages 1042-1079 (1974), because of the many variants and the complexity of the diazo method, the determination of bilirubin for a given sample is often quite different with different variants of the method. In addition, the diazo method can be time-consuming because it typically requires several reagents which must be freshly mixed for each determination. Moreover, the diazo method can be inaccurate because serum components other than bilirubin respond to diazotization.
In view of the foregoing, an enzyme preparation which specifically degrades bilirubin and which is therefore useful in assay compositions, elements, and methods represents a valuable contribution.